This invention relates to a method of modulating the immune response in a human, or other animal, in need thereof by inducing the production of interferon (IFN) by leukocytes which comprises administering an effective amount of influenza A NS1 to such human or animal; and to an immunomodulating pharmaceutical composition comprising an effective, leukocyte interferon production inducing amount of NS1 and a pharmaceutically acceptable carrier or diluent.
Several studies using virus systems have shown that virions, and in some instances isolated viral proteins, can enhance natural cytotoxicity. [See, e.g., Bishop et al., J. Immunol., 131, 1849 (1983); Casali et al., J. Exp. Med., 54, 840 (1981); Harfast et al., Scand. J. Immunol., 11, 391 (1985); Alsheikhly et al., Scand. J. Immunol., 17, 129 (1983).] Using the influenza virus glycoproteins (haemagglutinin and neuraminidase components), enhanced natural cytotoxicity of human peripheral blood mononuclear cells (PBMC) was demonstrated. [See, Arora et al., J. Virology, 52, 389 (1984).] This contrasts with recent observations that subunit influenza virus haemagglutinin, prepared by detergent solubilization, profoundly and irreversibly inhibits human natural cytotoxicity against K562 targets. [See, Ali et al., Immunol, 52, 687 (1984).] The results of these studies suggest that different molecular structures may mediate these different or diverse biological effects, although a more precise definition of the mechanisms involved is needed.
It has been shown that virus-infected target cells are extremely sensitive to NK-mediated lysis. This enhancement of lytic activity is thought to be mediated by endogenously produced IFN, but it is not established whether IFN from the infected target or the effector cell population is responsible for increasing cytotoxicity, although it is recognized that human NK cells can produce IFN upon appropriate stimulation. Human NK cells enriched by discontinuous Percoll density gradient separation can be stimulated by intact virus particles (influenza A and HSV-1, NDV and Sendai viruses) to release IFN, mainly IFN.alpha., although the production of IFN.gamma. has been observed with lymphocytes isolated from individuals seropositive for influenza A or CMV virus incubated with homologous viral antigen. [See, Djeu et al., J. Exp. Med., 156: 1222 (1982) and Starr et al., Infection and Immunity, 30: 17 (1980)]. In contrast, it was recently shown that detergent solubilized influenza virus haemagglutinin (HA) causes a profound and irreversible depression in human NK cytotoxicity. [See, Ali et al., Immunol., 52: 687 (1984)].
The function of NS1 and NS2 nonstructural proteins during influenza A viral infection is unclear. It is interesting to note that NS1 has been detected on the surface of virus infected cells [See, Shaw et al., J. Exp. Med., 156, 243 (1982)], and, it has been demonstrated serologically that extensive cross-reactivity exists between NS1 proteins from influenza A virus strains of human, avian, porcine and equine origin. [See, Shaw et al., cited above and Morrongiello et al., Intervirology, 8, 281 (1977).]
Young et al., "The Origin of Pandemic Influenza Viruses", W. G. Laver, editor, Elsevier Science Publishing Co., Inc. (1983), 129-137, review the cloning and expression of influenza virus genes and disclose the expression of the NS1 protein in bacteria cells (E. coli strain N99) transformed with a pAS1 expression vector containing the NS gene of influenza virus strain A/PR/8/34 (H1N1).
Young et al., Proc. Natl. Acad. Sci., U.S.A., 80, 6105-6109 (1983), disclose the expression of the NS1 protein by cells of E. coli strain N5151 transformed with a pAS1 expression vector containing the NS gene of influenza virus strain A/PR/8/34 (H1N1). Young et al. also disclose that the protein expressed by the NS gene was extracted, purified and injected into rabbits whose serum was subsequently used for immunoprecipitation and immunofluorescence assays.
Shaw et al., J. Exp. Medicine, 156, 243-254 (1982), disclose the purification of NS1 from cytoplasmic inclusions that were solubilized and used to raise antisera in rabbits; and also disclose that NS1 appeared to be highly conserved in different influenza A virus isolates. Shaw et al. state that since the NS1 antigen is expressed on the surface of infected cells, this suggests that an immune response to this protein could conceivably be of importance. Furthermore, Shaw et al. state that since there is extensive cross-reactivity in the NS1 proteins produced by different influenza A virus serotypes, NS1 related antigens should be considered as possible targets for cross-reactive cytotoxic T cells generated during infection.
Shaw et al., Infection and Immunity, 34(3), 1065-1067 (1981), disclose that the influenza A virus 23,000 dalton nonstructural protein, NS1, can be detected by direct immunofluorescence on the surfaces of infected mouse cells as early as 4 hours after infection with the A/WSN (H1N1) strain of influenza A virus. Shaw et al. conclude that since their results strongly suggest the surface expression of NS1 protein or a structurally related molecule on influenza A virus-infected cells, and since antigenic cross-reactivity has been shown for nonstructural antigens induced by different influenza A serotypes, NS1-related antigens should be considered as possible targets for cross-reactive cytotoxic T cells generated during influenza A virus infection.
Djeu, Clin. Immunol. Allerg., 3(3), 561-568 (1983), reviews the production of interferon by human natural killer (NK) cells and discloses that a large number of biological agents, including influenza virus strain A/PC, induce the production of interferon (IFN) by natural killer cells. Djeu also states that "since a vast array of biological agents can induce rapid IFN production by NK cells, it is tempting to speculate that the first step in defense (sic) against invading agents is the production of IFN which produces self-activation of NK activity in LGL (NK cells)."
Tiensiwakul et al., Intervirology, 20, 52-55 (1983), disclose that purified adenovirus fiber protein (FP) (a B-cell mitogen) induced the synthesis of interferon in murine cells.
SmithKline Beckman Corporation, European patent application Publication No. EPO, 176,493 Al, published Apr. 2, 1986, claims a vaccine for stimulating protection in animals against infection by influenza virus which comprises a polypeptide, other than an HA protein, having an immunogenic determinant of the HA2 subunit of an HA protein, wherein the immunogenic determinant is carried on a fusion protein having the N-terminal of the HA2 subunit fused to about 80 N-terminal amino acids of the NS1 protein which carries the HA2 subunit to assume an immunogenic configuration. SmithKline Beckman Corporation also disclose the cloning and expression of a coding sequence for the influenza A virus matrix protein.